1. Field of the Invention
The present invention relates to mutant vaccinia virus expression vectors. The mutant expression vectors of the present invention show substantially no virus replication in non-dividing cells and as such are superior to previous vaccinia virus expression vectors. More specifically, the vaccinia virus expression vectors of the present invention are associated with negative thymidine kinase and vaccinia virus growth factor phenotypes. The vaccinia virus vectors of the present invention are suitable for use as vaccines, cancer therapies, as well as for gene delivery vectors.
2. Description of the Related Art
Vaccinia virus has a long history of service as an expression vehicle. Vaccinia virus has been used most notably as a vaccination against smallpox, however, active immunization against smallpox is far less common since the eradication of the disease in 1977. Today, as a result of various advances in molecular biology, the vaccinia virus show promise as a vaccine against diseases other than smallpox.
The vaccinia virus genome can be manipulated using general molecular biology techniques well known in the art, to perform as an expression vector for exogenous, non-vaccinia virus genes. In one study, a region of the wild-type (WT) strain of vaccinia virus was shown to be suitable for the insertion and expression of foreign DNA. Mackett, et al., Proc Natl Acad Sci USA, 79(23):7415-9 (1982). The ability to express foreign DNA by exploiting a vaccinia virus vector created the possibility that vaccinia virus engineered to express exogenous, non-vaccinia virus genes could be used as gene expression vectors.
Recombinant vaccinia virus carrying exogenous nucleic acid encoding a pathogenic antigen protects an animal infected with the recombinant vaccinia virus and subsequently challenged with the pathogen from which the antigen was derived. For example, when live vaccinia virus recombinants expressing the hepatitis B virus surface antigen (HBsAg), the influenza A virus haemagglutinin, the herpes simplex virus (HSV) type 1 D glycoprotein, the rabies virus G glycoprotein, and the vesicular stomatitis virus G glycoprotein were used for immunization, animals were protected upon challenge with the pathogenic agent. However, post-immunization complications are documented concerning the use of vaccinia virus as a vaccine. One such concern involves the propensity of vaccinia virus to induce hyperplastic responses and even tumors in the skin of infected subjects.
The vaccinia virus growth factor (VGF) is thought to play a role in these complications. The VGF is a homologue of epidermal growth factor (EGF) and of transforming growth factor α. Blomquist, et al., Proc. Natl. Acad. Sci. USA, 81:7363-7367 (1984). Vaccinia virus growth factor that has been post-translationally modified is capable of binding to the EGF receptor, stimulating autophosphorylation and inducing anchorage-independent cell growth.
To investigate the role of this gene product on infectivity and viral yields, both copies of the VGF gene were deleted from a recombinant vaccinia virus. In tissue culture cells there was little or no difference in infectivity or yield between the wild-type and mutant viruses. Buller, et al., J. Virol. 62:866-874 (1988). In vivo experiments revealed, however, that infection with wild-type virus resulted in a rapid proliferation of extodermal and entodermal cells of the chicken embryo chorioallantoic membrane, whereas this did not occur to the same extent with the VGF double mutant. Buller, et al., Virology, 164:182-192 (1988). However, there was double mutant viral reproduction in these experiments.
Another line of research examined the role of the thymidine kinase (TK) gene in vaccinia virus reproduction. It is known that the TK gene enhances herpesvirus replication in non-dividing cells. Kiemperer, et al., Virology 31:120-128 (1967). To determine whether inactivation of the vaccinia virus TK gene by the insertion of foreign DNA led to attenuation, Buller, et al., examined several TK− recombinants for their ability to replicate. Buller, et al., Nature 317:813-815 (1985). Although reduced viral mediated pathogenesis was observed with these mutants, the TK− viruses retained the ability to replicate in non-dividing cells.
Although the vaccinia virus has been the subject of considerable research to date, there remains a need for a vaccinia virus expression vector that substantially fails to replicate in non-dividing cells.